Derivatization of polymeric supports is central to the preparation of various types of diagnostic and chromatographic media. Coupling of ligands to supports, i.e. covalent attachment of specific molecules or functional groups, is necessary to impart to those supports the ability to effect the separation, identification, and/or purification of molecules of interest. Prior art techniques for controlling the concentration or density of ligands on a polymeric support generally fall into one of four categories or combinations thereof:
a) Manipulation of reaction conditions which "activate" the matrix, i.e. which introduce a reactive group which can couple to the ligand. This often involves varying the concentration of "activating reagents", reaction time, reaction temperature, pH, or combinations of these variables.
b) Manipulation of reaction conditions during coupling of the ligand to the support. This may involve varying the concentration and/or the total amount of ligand the support is challenged with, ionic strength of the coupling buffer, and type of salt in the coupling buffer as well as the variables of time, temperature, pH, etc., mentioned above.
c) Manipulation of the amount of reactive or "activatable" functional group incorporated into the polymer support by varying polymer composition at the time of its formation, i.e. during the polymerization.
d) Manipulation of the amount of ligand incorporated into the polymer by preparation of a polymerizable ligand monomer and varying the concentration of this monomer in the monomer feed during polymerization.
For the most part, the above techniques for controlling ligand concentration on polymeric supports are quite difficult to apply in a practical and reproducible manner, primarily because of the large number of variables which must be simultaneously controlled. This is especially true of the first two techniques, in which the efficiencies of the reactions (i.e., extent of desired reaction as opposed to competing side reactions) is strongly influenced by reaction conditions. Technique "c" seems to offer some degree of control, although one must subsequently apply the techniques of "a" and/or "b" in a second step to couple the ligand. Technique "d" would appear to provide exact control of ligand density until one realizes that many of the ligands which are useful for diagnostic applications and chromatographic separations contain functional groups which are incompatible with conditions necessary for formation of the desired polymer (e.g., they are unstable under the contemplated polymerization conditions, or they interfere with the polymerization reaction, such as by inhibition of polymerization).
Successful ligand coupling is based on two factors: quantity immobilized and quality of immobilization. Quantity immobilized, expressed as weight of ligand per unit volume of support, is an indicator of the amount of ligand coupled regardless of the quality of that immobilization. Quality of immobilization is an indicator of the relationship between the amount of ligand coupled onto a support and the ability of that ligand to maintain its binding interactions with a ligate such that it retains usefulness for chromatographic or diagnostic activity. Optimizing that activity is desirable and can be accomplished by manipulating either quantity or quality of immobilization, or both, depending on the desired use application. However, there must be enough ligand density to achieve practical utility.
While a support can couple at maximum ligand density, the binding of ligate to that ligand during chromatography can be hindered or altered by a number of factors including multiple-site binding of the ligand to the support (especially relevant with high molecular weight ligands) and steric hindrance due to proximity of adjacent ligands. Thus, overly-dense ligand coupling to a support is wasteful of the ligand and unnecessary or deleterious to the binding activity, especially for such applications as affinity chromatography and diagnostics. In these instances, the optimal condition in ligand coupling would be the achievement of maximum possible density of ligand coupled while maintaining maximum chromatographic or diagnostic activity with respect to ligate binding to said ligand. That results in optimal ligate binding or functional efficiency of the coupled ligand on the support.
For other chromatographic separations, such as for hydrophobic interaction or reverse phase, chiral, and ion exchange chromatography, high quantity of immobilization may lead to too strong a binding interaction, thus leading to difficult elutions or losses in resolution. In these cases, the ability to decrease ligand density or alter its distribution will improve chromatographic performance in terms of selectivity, resolution, and recovery of ligate.
Many ligand candidates are large molecules such as proteins and enzymes that have specific conformations necessary to retain biological activity. Recently attempts have been made to overcome the limitations in the prior art with respect to coupling of these high molecular weight ligands. In the case of antibody binding of antigen, low antigen binding efficiencies have been attributed to the concerted actions of surface density of antibody, multi-point attachment of antibody to porous supports, and undesirably restrictive conformations imposed by covalent attachment. See Velander et at., "The Use of Fab-Masking Antigens to Enhance the Activity of Immobilized Antibodies", Biotechnology and Bioengineering, Vol. 39, 1013-1023 (1992) which discloses how enhanced functional efficiency was achieved when the Fab portion of a monoclonal antibody was masked with synthetic antigens prior to covalent immobilization of the antibody on the support, followed by unmasking.
U.S. Pat. No. 5,200,471 (Coleman et al.) describes a method for the covalent immobilization of biomolecules on azlactone-functional polymeric supports in the presence of polyanionic salt and a buffered aqueous solution. Preferably, the immobilization also occurs in the presence of an azlactone quencher, i.e. another molecule which will compete with the protein ligand for the azlactone groups through which covalent coupling occurs. Incorporation of the quencher in this procedure in concentrations in the range of 4 to 6 orders of magnitude higher than the concentration of the biomolecule seems to have some effect on controlling the density of the coupled protein ligand. However, its major effect appears to be manifest in improvements in bound specific biological activity of the ligand.
Other attempts to control ligand density and/or distribution of biomolecules are described in U.S. patent application Ser. No. 08/038,645 (Velander et al.) and in references therein.
Using a very different approach, U.S. Pat. No. 4,968,742 (Lewis et al.) teaches a two step process for control of ligand density by controlling the number of active sites for ligand coupling. Step one of the complicated process involves reacting the non-activated polymeric material with a predetermined ratio of excess amounts of an "activating" agent and a "blocking" agent. In a second step, the ligand without any other agent competing for coupling sites is covalently coupled to a functional group on the "activating" agent. While this method again offers some degree of control over the density of ligands, it is still somewhat limited. The method indeed only allows control over the introduction of the "activating" agent, with the, subsequent coupling of the ligand being subject to the deficiencies discussed above.